2.3.1. Library Preparation

Purified genomic DNA from each sample was initially amplified in a 25 μL reaction, using the REDExtract-N-Amp PCR ReadyMix (Sigma-Aldrich, St Louis, MO, USA) with 0.4 μM of each 16S rRNA gene primer and 2 μL template. The 16S PCR conditions were the following: an initial denaturation at 95 ◦C for 2 min, 20 cycles of 95 ◦C for 30 s, 60 ◦C for 1 min and 72 ◦C for 30 s and final elongation at 72 ◦C for 7 min. This PCR run is referred to as PCR1. The product from PCR1 was prepared for sequencing by a second PCR (referred to as PCR2), using the same PCR protocol as described above. PCR2 attached an adaptor A, an index i5 and a forward sequencing primer site (FSP) in the 5' end of the amplicons and an adaptor B, an index i7 and a reverse sequencing primer site (RSP) to the 3' end of the amplicons. DNA was quantified using the Quant-ITTM dsDNA High Sensitive Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and PCR2 products were pooled in equimolar amounts between samples. Agencourt AMPure XP bead (Beckman Coulter, Brea, CA, USA) purification was performed to remove undesirable DNA amplicons from the pooled amplicon library (PAL) in a two-step process. First, DNA fragments below 300 nucleotides length were removed by a PAL AMPure beads 10:24 ratio, following the manufacturer's instructions, and eluted in 40 μL TE bu ffer (AM1). Secondly, large DNA fragments above 1kbp were removed by AM1 to AMPure beads 10:16 ratio as previously described. The resulting AMPure beads purified PAL (bPAL) was diluted to a final concentration of 11.5 pM DNA in a 0.001 N NaOH and used for sequencing on the Illumina MiSeq desktop sequencer (Illumina Inc., San Diego, CA, USA). The library was sequenced with the 500-cycle MiSeq Reagent Kit V2 in a 2 × 250 nt setup (Illumina Inc., San Diego, CA, USA). The sequencing was performed at Statens Serum Institute (SSI).
